Model for hematopoietic cell growth, differentiation and desease

ABSTRACT

The invention is a genetic construct comprising the coding sequence for a hormone dependent E2a-Pbx1 protein. The invention is the use of the construct to generate hematopoietic cell lines arrested in differentiation upon exposure to the appropriate hormone. Upon removal of the hormone, cells undergo normal, synchronous differentiation providing a system for the study of normal differentiation. Expression of a number of oncogenes in the cells maintains the cells in an undifferentiated state, providing a model for the study the mechanisms of leukemia, and for the testing of pharmacological agents for the treatment and amelioration of the disease.

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S.provisional application Serial No. 60/250,631 filed Nov. 30, 2000 whichis incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Molecular pathways of normal hematopoietic cell differentiation,as well as the mechanisms by which oncogenes disrupt this process,remain poorly understood. In normal hematopoietic progenitor cells, aprogram of specific gene expression orchestrates commitment anddifferentiation of mature cells to multiple different lineages. In acuteleukemias, however, oncoproteins interfere with this genetic program,resulting in the unregulated proliferation of cells that no longerretain the capacity to differentiate normally. In acute myeloidleukemias (AMLs) many known myeloid oncoproteins can block thedifferentiation of normal progenitors cultured in vitro in the presenceof granulocyte-macrophage colony stimulating factor (GM-CSF) orinterleukin-3 (IL-3). However, neither the genetic events that underlienormal hematopoietic cell differentiation nor the mechanism throughwhich leukemic oncoproteins interfere with the execution of the programof lineage differentiation are well understood.

[0003] A number of genes have been identified that are criticallyinvolved in various forms of leukemia. For example, the t(1;19)chromosomal translocation in humans results in the production ofE2a-Pbx1, a chimeric oncoprotein containing the transactivation domainsof E2a joined to the DNA-binding homeodomain protein Pbx1. E2a-Pbx1causes T-cell and myeloid leukemia in mice, blocks differentiation inmurine myeloid progenitor cells, and transforms fibroblasts. Themechanisms of differentiation arrest are likely accompanied by aberrantexpression of tissue specific and developmentally regulated genes. Thisaberrant tissue specific gene expression is also found in the subset ofpre-B cells containing the t(1:19) translocation in humans. The exactmechanism by which E2a-Pbx1 alters gene expression is unclear, but itappears to modulate transcription in cooperation with homoebox geneproducts. In human pre B-ALL, expression of E2a-Pbx1 correlates with theexpression of EB-1, a tyrosine kinase signal transduction gene.Potentially, EB-1 overexpression could interfere with normal signaltransduction pathways in proliferation and differentiation.

[0004] Primary cells and myeloid cell lines offer useful, but limited,models to approach questions regarding mechanisms of normal myeloid cellgrowth and differentiation, and how this process goes awry in leukemia.Although primary marrow progenitor cells demonstrate normal granulocyticand monocytic differentiation in IL-3 or GM-CSF, one is limited by thescarcity of cells, the difficulty in isolating homogeneous populations,and the inability to verify expression of non-transforming oncoproteinswhen using such progenitor cells to study the normal program of myeloiddifferentiation and the mechanisms by which oncogenes alter thisprogram. Useful myeloid cell lines that demonstrate inducibledifferentiation in response to changes in cytokines include FDCPmixA4(GM-CSF+granulocyte colony-stimulating factor [G-CSF]+macrophagecolony-stimulating factor [M-CSF]), 32Dlc3 (G-CSF), M1-AML (IL-6), andFDB cells (GM-CSF), whereas those that respond to nonphysiologic stimuliinclude HL60 (high levels of retinoic acid [RA],12-o-tetradecanoylphorbal 13-acetate [TPA], dimethyl sulfoxide [DMSO]),EML (GM-CSF and RA), MPRO (RA), NB4 (RA), and U937 cells (RA, TPA, DMSO,or vitamin D₃). There are no lymphoid cell lines that demonstrateinducible differentiation. Although these lines supply an unlimitednumber of clonal cells, most are limited by the fact that they containundefined genetic changes such that their differentiation is oftenincomplete, asynchronous, or accompanied by cell death. Because themyeloid cells are already blocked to differentiation in response toeither IL-3 or GM-CSF, it is unclear whether induction by otherextrinsic factors proceeds through normal differentiation pathways.Furthermore, oncoproteins whose action it is to block differentiationinduced by IL-3 or GM-CSF cannot be assayed in these prearrested celllines.

[0005] A murine cell line was derived by Hogg and coworkers using ac-Myb-ER fusion was described. However, clonal c-Myb-ER cell lines werenot derived, nor were the cells assayed for their ability to scoredifferentiation arrest by other oncoproteins (Hogg et al. (1997)Oncogene 15:2885-98). Thus, the cell line overcomes some of the problemsassociated with established cell lines; however, the cell line is notclonal. This results in problems with reproducibility and maintenance ofexpression constructs. It is unclear that such a system would serve as agood model for myelopoiesis or AML.

[0006] An optimal hematopoietic cell line model would (1) lackconstitutive expression of interfering oncoproteins, (2) exhibitconditional and terminal differentiation in response to biologicallyrelevant molecules such as interleukins and growth factors, and (3) beblocked in an undifferentiated state by common leukemic oncoproteins.None of the cell lines listed above meet all of these criteria.

SUMMARY OF THE INVENTION

[0007] The invention is a chimeric cDNA molecule encoding a conditionalE2a-Pbx1 protein which contains a hormone binding domain (HBD) from ahormone receptor including, but not limited to, estrogen receptor (ER),glucocorticoid receptor (GR), thyroid hormone receptor (THR),mineralocorticoid receptor (MR), androgen receptor (AR), andprogesterone receptor (PR). The invention is also the protein expressedfrom the cDNA of the invention. When expressed in the cell in theabsence of hormone, it is thought that the chimeric protein is masked byheat shock proteins (HSPs) and is prevented from binding to the cellularDNA. Upon binding of the hormone to its HBD, the chimeric protein isreleased from the HSP and is free to bind to the cellular DNA activatingtranscription.

[0008] The invention is a primary hematopoietic cell line expressing thecDNA of the invention. The fate of the cell line is dependent upon thesource of the cells and the interleukins and growth factors to whichthey are exposed. For example, bone marrow, fetal liver and embryonicstem cells can be induced to become myeloid cells in the presence ofGM-CSF or IL-3. In the presence of IL-7, the same cells will be inducedto become lymphocytes. Expression of the construct of the invention inthese cells in the presence of hormone results in a differentiationblock in the cells. Upon removal of hormone, the cells undergo normal,terminal, synchronous differentiation. However, upon expression of aheterologous leukemic oncoprotein, the cells remain blocked in theirundifferentiated state, paralleling the situation seen in leukemia.

[0009] The invention is the use of the cell lines to examine thebiochemical and genetic pathways that accompany normal differentiationin hematopoietic cells, as well as a system in which to dissect howother leukemic oncoproteins interfere with these pathways. Presentlythere is no conditional model of differentiation arrest thatrecapitulates the process seen in leukemia in vivo. Terminallydifferentiated cells do not divide; therefore, the maintenance of cellsin an undifferentiated state is required for the uncontrolled celldivision associated with cancer. The cell lines of the system allow fordetailed analysis of the mechanisms of the inhibition of differentiationassociated with leukemia as established by E2a-Pbx1 or other singleoncoprotein.

[0010] The invention provides a system for the development and testingof pharmacological agents to develop methods to ameliorate and cureleukemias. By understanding the mechanisms of leukemogenesis, one candesign agents to disrupt the mechanisms. In an assay to test forchemotherapeutic activity, the cell lines of the invention are grown inthe presence of the appropriate hormone to maintain the cells in aproliferative state. Alternatively, the cells of the inventionexpressing the conditional E2a-Pbx1 are subjected to another factor(e.g. introduction of a leukemic oncoprotein) that prevents the normaldifferentiation of the cells in the absence of hormone. Cells aresubsequently treated with any of a variety of factors including, but notlimited to compounds from combinatorial libraries, developed by rationaldrug design, or purified from natural products, or genetic constructs inappropriate delivery vectors. In this manner, one can analyze the cellsto identify factors that are capable of inducing the cells todifferentiate, thereby arresting their unregulated cell division.

[0011] The invention is a system for the study of other oncogenesinvolved in the process of hematopoiesis. The transformed primary celllines of the instant invention are defined and contain only theconditionally activated E2a-Pbx1-HBD, rather than unknown changes thatmay exist in cell lines. Thus the effect of the introduction of theoncogene of interest can be determined without interference from otherunknown oncogenes or cellular changes. The cooperative effects ofoncogenes such as Bcl-2, Ras and Bcr/Abl with E2a-Pbx1 in the process ofdifferentiation and leukogenesis can also be analyzed. The cell linescan also be used to assess the oncogenic potential of various factorsincluding nucleic acids, proteins, growth factors and other bioactivecompounds by their ability to maintain differentiation arrest after thewithdrawal of hormone.

[0012] The invention is a system that allows for the identification ofE2a-Pbx1 target genes that mediate the differentiation block as theblock can be turned on and off by the addition or removal of hormone.Cells expressing either form of the E2a-Pbx1-HBD constructs can becompared to cells before and after withdrawal of the appropriatehormone, or with cells expressing any of a number of oncoproteins inconjunction with E2a-Pbx1-HBD. Analysis may be performed by any of anumber of methods well known to those skilled in the art.

[0013] The invention is a system to identify differences between cellfates by providing cell lines that are arrested in cellulardifferentiation, but not lineage definition. The cells can be releasedfrom a differentiation block in a synchronous manner such that asufficiently large homogenous population of cells can be obtained forstudy. Analysis of a variety of factors, including but not limited totranscription factors, cell surface markers, protein activation throughphosphorylation or other mechanisms, translocation of proteins withinthe cell, activation of promoter, enhancer and repressor elements, andRNA transcription can be performed by a variety of methods well known tothose skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1. Estrogen dependent forms of human E2a-Pbx1 are produced byreplacement of Pbx sequences with the ER HBD. EP Δ578ER and EP Δ623ERwere created by an internal fusion of the HBD (aa 282-595) of theGly400Val mutant human ER. The HBD replaces the Pbx1 sequences, upstreamof the DNA-binding homeodomain, that are dispensable for the biochemicaland transforming properties of E2a-Pbx1.

[0015] The present invention will be better understood from thefollowing detailed description of an exemplary embodiment of theinvention, taken in conjunction with the accompanying drawings in whichlike reference numerals refer to like parts and in which:

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

[0016] The molecular mechanisms of hematopoietic cell growth anddifferentiation are not well understood. In normal progenitor cells,differentiation is directed by exposure to growth factors andinterleukins directing cells to a specific, terminal and quiescent fate.In acute leukemia, progenitor cells fail to respond normally todifferentiation inducing signals and divide uncontrollably. Theinvention is a cDNA that encodes a protein that acts as a molecularswitch to induce a block in differentiation by activation of an E2a-Pbx1protein. The protein contains a hormone binding domain that allows forthe activation of the protein in the presence of hormone. Upon removalof the hormone, the progenitor cells undergo a normal, terminal andsynchronous process of differentiation. Thus the cells provide an idealmodel for the process of hematopoiesis. However, upon expression of aleukemic oncoprotein in the E2a-Pbx1-ER cells, followed by removal ofhormone, the differentiation block is maintained, providing an idealmodel for leukemia.

[0017] A model of myeloid cell differentiation was established usingmouse primary bone marrow cells differentiated in the presence of GM-CSFand an E2a-Pbx1 protein containing the HBD from the estrogen receptor(E2a-Pbx1-ER). Clonal cell lines expressing E2a-Pbx1-ER were blocked inan undifferentiated state in the presence of estrogen, butdifferentiated normally into granulocytes and monocytes upon removal ofestrogen. However, upon expression of any of a number of oncoproteins inthe cell, including AML1/ETO, PML/RAR α, Hoxa9, Hoxb8and wild typeE2a-Pbx1, the differentiation block was maintained as in AML.

[0018] The myeloid clonal cell line expressing E2a-Pbx1-ER described indetail below is an example of the cell lines that can be derived usingthe method of the invention. The selection of the source of the primarycells is a matter of choice and not a limiting aspect of the invention.Bone marrow, fetal liver and embryonic stem cells from a number ofmammalian sources can all be induced to adopt a myeloid fate uponexposure to GM-CSF and IL-3. The same cells can be induced to lymphoiddifferentiation by exposure to IL-7. Similarly, the HBD selected is amatter of choice. The selection of the HBD is dependent on the effectthat the ligand will have on the cell line, preferably none, and theavailability of reagents. Such selections are routinely made by thoseskilled in the art (Mattioni et al. (1994) Meth in Cell Bio. 43:335-352,incorporated herein by reference). The appropriate hormone is defined asthe hormone that binds the receptor in vivo, e.g. progesterone for theprogesterone receptor and glucocorticoid for the glucocorticoidreceptor. The appropriate hormone is also defined as any analog,agonist, antagonist or other compound that binds to the hormone bindingdomain in a specific manner to activate the conditional E2a-Pbx1 fusionprotein.

[0019] The detailed analysis below describes the use of murine myeloidprecursor cells infected with a retrovirus containing the codingsequence of human E2a-Pbx1 fused to the HBD of the human ER. Theproteins and genetic elements tested in the system are also derived fromboth mouse and human demonstrating that there is efficient cross-speciesinteraction. This is not surprising as it is well known that manyproteins are well conserved throughout evolution, and that humanproteins have been shown to function in species as disparate as mouseand even yeast. Thus, it possible to assay proteins and genetic elementsacross species in the cell lines of the instant invention.

[0020] The invention is not limited by the selection of the use of aspecific HBD such as that from the ER in the specific example. Moreoverit is not limited by the selection of a compound to bind the ER-HBD. Anyestrogen, estrogen analog (e.g. β-estradiol, 4-hydroxytamoxifen) oragonist can be used to activate the E2a-Pbx1-ER fusion protein. Forsimplicity, the term estrogen is used throughout the application. Itshould be understood to mean any compound capable of activating thefusion protein in a specific manner.

[0021] E2a-Pbx1 is the result of a t(1:19) translocation that joins thetransactivation domains of E2a with the DNA-binding homeodomain of Pbx1.A conditional E2a-Pbx1 (E2a-Pbx1-ER) was constructed by insertion of theligand binding domain of the estrogen receptor between the E2a and thePbx1 domains (FIG. 1), creating a protein that is constitutivelyexpressed, but functionally inactive in the absence of estrogen,estrogen analogs (e.g. estradiol) or other agonist or antagonist. Fusionof the ER HBD to the — or C-terminus of the full-length E2a-Pbx1produced proteins that were estrogen-dependent at the level oftranscription, but were unstable (expressed only 10-30% of wild-type)and failed to exhibit estrogen-dependent transformation. Therefore, Pbx1sequences N-terminal to the homeodomain, which are dispensable for thebiochemical and transforming properties of E2a-Pbx1, were replaced withthe ER HBD (as detailed in Example 1), creating EP Δ578ER and EP Δ623ERproteins (FIG. 1). Any amount of the Pbx1 domain of the E2a-Pbx1 proteinmay be deleted between amino acids 487 and 623 and replaced with the HBDof choice. It is not necessary to delete any of the PBx1 sequence. EPΔ578ER and EP Δ623ER are stable, constitutively expressed, anddemonstrate estrogen-dependent biochemical and oncogenic functions.

[0022] E2a-Pbx1-ER demonstrated estrogen-dependent activation onartificial luciferase reporter constructs in cells that endogenouslycontain high and low levels of Hox proteins, namely NIH 3T3 cells andNalm-6-pre-B cells, respectively. Hox-dependent cooperativetransactivation was additionally assayed by co-transfection ofE2a-Pbx1with either Hoxc8 or Hoxa9 constructs into Nalm-6 cells with theluciferase reporter construct. Collectively, the strikingly similarpatterns of transactivation of luciferase expression in theseexperiments demonstrated that the activation by wild-type E2a-Pbx1 isunaffected by estrogen, and that EP Δ578ER and EP Δ623ER participate inboth Hox-dependent and Hox-independent transcriptional activation in astrictly estrogen dependent manner. The weaker transactivation ofluciferase expression of EP Δ578ER, as compared to EP Δ623ER, parallelsthe weaker cooperativity of the parental EP Δ578 protein with Hoxpartners in both DNA-binding and transactivation assays. Estrogen(β-estradiol at 1 μM) was sufficient for inducing maximaltransactivation, and estrogen induced luciferase activity could bedetected within 45 minutes of the addition of estrogen, with maximalactivity attained at 8 hours.

[0023] The E2a-Pbx1-ER constructs were able to induce conditional andreversible proliferation of fibroblasts in a manner similar to wild-typeE2a-Pbx1. NIH 3T3 cell lines stably expressing wild-type E2a-Pbx1, EPΔ578ER and EP Δ623ER were established. In density dependentproliferation assays (see Example 2), wild-type E2a-Pbx1 induced anapproximately 3.5 fold increase in total cell number as compared tocells carrying vector alone. In the absence of estrogen, neither EPΔ578ER nor EP Δ623ER stimulated cell proliferation in the absence ofestrogen, whereas the addition of estrogen induced a 1.2-fold and4.5-fold increase in cell number, respectively. This is consistent withthe relative transforming strength of EP Δ578ER and EP Δ623ER proteins.Upon removal of estrogen, cells conditionally transformed by EP Δ578ERor EP Δ623ER reverted to the growth densities exhibited by the parentalNIH 3T3 fibroblasts, demonstrating that the transformation induced by EPΔ578ER and EP Δ623ER if fully reversible.

[0024] Estrogen-dependent immortalization of primary murine marrowGM-CSF-dependent myeloid progenitor cells was observed upon infectionwith EP Δ578ER and EP Δ623ER. Both EP Δ578ER (12 of 12 cultures) and EPΔ623ER (6 of 6 cultures) immortalized progenitor cells as efficiently asE2a-Pbx1, but only in the presence of 1 μM estrogen. Wild-typeE2a-Pbx1-, EP Δ578ER-, and EP Δ623ER-immortalized progenitor cells werephenotypically identical. All cell lines were dependent on GM-CSF, whichcould be substituted with IL-3. The cells were unresponsive togranulocyte (G)-CSF, or to macrophage (M)-CSF, undergoing apoptotic celldeath within 24 hours when cultured in these or in the absence ofcytokines.

[0025] Removal of estrogen from polyclonal populations of Δ578ER- or EPΔ623ER-immortalized myeloblasts evoked synchronous morphologicdifferentiation to mature neutrophils (about 80%) characterized bysegmented nuclei and lightly staining cytoplasm, and to maturemacrophages (about 20%) characterized by their larger size and ovalnuclei. Granulocytes were fully differentiated within 5 to 6 days andsurvived for an additional 24 to 48 hours, whereas monocytes wereadherent and stopped proliferating after 7 to 9 days.

[0026] The expression of cell surface markers on one population of EPΔ578ER-immortalized cells was characterized by flow cytometry. In thepresence of estrogen, cells did not stain with GR-1 (lipopolysaccharidereceptor Ly6G) or F4/80 and stained weakly with Mac-1 (CD11b). Theremoval of estrogen resulted in the dramatic up-regulation of thesemyeloid differentiation markers, paralleling the morphologicdifferentiation to granulocytes and monocytes. Functional NADPH oxidaseactivity, indicative of myeloid differentiation, as evidenced by darkblue deposits following the NBT reduction assay, was observed in fewerthan 1% of the progenitor cells in the presence of estrogen, and in morethan 99% of the cells cultured 7 days in the absence of estrogen.

[0027] Expression of cell surface markers was accompanied bytranscriptional activation of a number of differentiation markers. Thisis not observed in all models of myeloid differentiation. In somesystems, phenotypic changes are not accompanied by normal expression ofterminal differentiation markers, making the systems inadequate for thestudy of the process of differentiation. Northern blot analysis wasperformed on a 9-day time course of RNA samples collected from EPΔ578ER-immortalized cells differentiated in the absence of estrogen. Theexpression of genes encoding the master transcriptional regulators,primary and secondary granule proteins, components of the NADPH oxidasecomplex and other markers of myeloid differentiation was examined.

[0028] In both the presence and the absence estrogen, progenitor cellsdemonstrated stable expression of the Ets-family member PU.1, as well asmembers of the CCAAT/enhancer binding protein (c/EPB α, β, and ε) familyof transcription factors, all of which have been shown to be essentialfor normal myeloid development. C-Myb and c-Myc showed characteristicdownregulation late in differentiation, whereas AML1 showed marked(about 5-fold) up-regulation following the removal of estrogen.

[0029] The sequential expression of primary and secondary granule genesalso accompanied differentiation. Primary granule genes neutrophilelastase and (NE) and myeloperoxidase (MPO) were rapidly upregulatedfollowing estrogen withdrawal. This rapid upregulation is important inthe context of elucidating differentiation arrest by E2a-Pbx1 byproviding a means to identify transcriptional activators within the NEand MPO promoters, of transcriptional activators that are not produced,or transcriptional repressors that are not eliminated in the presence ofactive E2a-Pbx1. Such genetic defects provide molecular handles toidentify the direct E2a-Pbx1 target genes that mediate differentiationarrest.

[0030] Secondary granule genes lactoferrin (LF) and neutrophilgelatinase (NG) were not expressed in myeloblasts and were activated atday 2 and day 7 of differentiation, respectively. Examination of the twosubunits of the NADPH oxidase complex revealed that p₄₇ ^(PHOX) wasconstitutively expressed, whereas gp91^(PHOX) was not expressed untilday 4 of differentiation. The regulated expression of gp91^(PHOX) isconsistent with the timing of robust NADPH oxidase activity asdetermined by NBT reduction. The cells also demonstrated transcriptionalupregulation of genes encoding cell surface receptors Ly6G (GR-1), CD14and the G-CSF receptor. Ly6G expression paralleled that seen by flowcytometric analysis and the low level of G-CSF-R expression inprogenitor cells is consistent with their G-CSF unresponsiveness.

[0031] Conditional immortalization by EP Δ578ER and EP Δ623ER allowedfor the examination of whether all progenitors seemingly identical bymorphological criteria, were committed to identical patterns ofdifferentiation. Populations of progenitor cells were cloned, clonalitywas confirmed by retroviral integration analysis using Southernblotting, and phenotypes were examined using light microscopy andWright-Giemsa staining 5 days after removal of estrogen. Three types ofclones exhibiting E2a-Pbx1-mediated conditional myeloid differentiationwere identified: bipotential clones that differentiated into bothgranulocytes and monocytes (ECoM-GM), clones exhibiting restrictedgranulocytic differentiation (ECoM-G), and one clone exhibitingrestricted monocytic differentiation (ECoM-M).

[0032] Within 4 days, ECoM-G cells differentiated homogeneously togranulocytes, whereas ECoM-M cells required 7 days for quantitativemonocytic differentiation. Both ECoM-G and ECoM-M cells acquiredfunctional NADPH oxidase activity and the ECoM-M cells upregulatednonspecific esterase (NSE), a characteristic marker of normal macrophagedevelopment. The ECoM-G cells showed dramatic increase in GR-1 (Ly6G)and Mac-1 staining 7 days after estrogen withdrawal, whereas theintensity of the F4/80 staining was significantly reduced. In contrast,the ECoM-M cells did not express GR-1, but were positive for and showedincreasing staining of both Mac-1 and F4/80 during monocyticdifferentiation. Both ECoM-G and ECoM-M cells became functionallyphagocytic as demonstrated by the ability to engulf FITC-labeled E. coliBioParticles (see Example 4).

[0033] Phenotypic changes following the removal of estrogen wereaccompanied by reduced proliferation and G₁-cell cycle arrest asevidenced by DNA content analysis. Although both ECoM-G and ECoM-Mprogenitor cells in estrogen had a high S-phase fraction (G₁ 48%/G₂ 8%/S44% and G₁ 35%/G₂ 13%/S 52%), the majority of the cells had accumulatedin G₁ following 7 days of differentiation in the absence of estrogen (G₁96%/G₂ 2%/S 2% and G₁ 83%/G₂ 11%/S 6%).

[0034] The parallel models of granulocyte and monocyte differentiationallow for the examination of lineage specific gene expression. ECoM-Gand ECoM-M cells recapitulated well established patterns of myeloid geneexpression including the down-regulation of c-Myb and c-Myc, and theupregulation of Egr-1, microsialin, c-Fos, neutrophil collagenase (NC),c-Fms and macrophage scavenger receptor SRA-1. Zinc finger protein andtranscriptional repressor Gfi-1 was down-regulated in both ECoM-G andECoM-M clones, whereas expression of family member growth factorindependent (Gfi)-1 was down-regulated in only the ECoM-M clone, similarto a previous report in which its down-regulation accompanied thecell-cycle arrest, and up-regulation of p21^(WAF/CIP1) during IL-6induced monocytic differentiation of M1-AML cells. Ear-2 was stablyexpressed throughout the differentiation of both ECoM-G and ECoM-M cellsin contrast to a previous report in which it was shown to bedown-regulated during the G-CSF-induced granulocytic differentiation in32Dcl3 cells and hypothesized to bind and inhibit AML1 activity.Similarly, Ets family member Fli-1 has been reported in human T-cell,B-cell and myeloid leukemia lines and was shown to be critical forhematopoiesis based on a study in a Fli-1 deficient mouse. Expression ofthe aldoketoreductase mAKRa was restricted to the ECoM-G clone and wasstable throughout differentiation, in contrast to a previous report thatshowed decreased expression following the all-trans-RA (atRA)-inducedgranulocytic differentiation of EML-C1 and MPRO cells. Expression ofEts-2 was restricted to the ECoM-G clone, which was unexpected givenprevious reports of Ets-2 expression in normal and transformedmacrophages.

[0035] Overall, the differences in gene expression between ECoM-G andECoM-M cells demonstrate that E2a-Pbx1 prevents differentiation but notlineage definition, and that these cell lines are useful models toidentify differences between granulopoiesis and monopoiesis.

[0036] The cell lines of the invention are also a model system in whichto study the inhibition of differentiation arrest due to the expressionof a heterologous oncoprotein. A variety of oncogenes were tested fortheir ability to maintain the differentiation arrest in the E2a-Pbx1-ERcell lines upon withdrawal of estrogen (Examples 6-9). Expression of anumber of heterologous oncogenes were able to maintain the cells in astate of differentiation arrest. However, the specific markers expressedin the cells varied depending on the cooperative oncogene expressed.Such cells provide an ideal model for the understanding leukemia as anyof a number of cooperative mutations are possible in vivo. Determinationof the cooperative mutation by the analysis of markers from patientsamples can lead to more efficient therapies for the treatment ofdisease.

[0037] The invention also provides a system for the testing of variouspharmacological compounds and therapeutic reagents for the treatment ofdisease. The ability of a therapeutic agent to promote differentiationinduced by a variety of single oncoproteins in a defined backgroundcould only be studied using the system of the instant invention.Expression of a heterologous oncogenes in a cell line of the inventionallows for the direct comparison of the efficacy of a single compound oncells with identical genetic backgrounds other than the single oncogene.Alternatively, a series of compounds can be tested on cells containing adefined genetic alteration to determine if they are capable of inducingdifferentiation.

EXAMPLE 1

[0038] Construction of estrogen-dependent versions of E2a-Pbx1. In humant(1:19) pre-B ALL, both E2a-Pbx1a (825 amino acids (aa)) and E2a-Pbx1b(742 aa) are expressed, differing only in residues C-terminal to thePbx1 homeodomain. Sequences encoding the HBD of the ER, residues 282 to595 of the Gly400Val human ER, with flanking MluI restriction sites,were amplified by high-fidelity polymerase chain reaction (PCR). TheGly400Val mutant ER was used because the point mutation renders thereceptor insensitive to the low levels of estrogen found in fetal bovineserum (FBS) as well as to the estrogenic effects of compounds such asphenol red. The estrogen-binding domain was inserted into two versionsof E2a-Pbx1b (E2a-Pbx1 Δ487-578 and E2a-Pbx1 Δ487-623) that lack Pbx1sequences N-terminal to the Pbx1 homeodomain (FIG. 1) that have uniqueMluI sites in place of the deleted residues, resulting in fusionsdesignated EP Δ578ER and EP Δ623ER, respectively. Thus, the HBD replacesPbx1 sequences upstream of the DNA-binding homeodomain that aredispensable for the biochemical and transforming properties of E2a-Pbx1.EP Δ578ER and EP Δ623ER were subcloned into the murine stem cell virus(MSCV) retroviral vectors MSCVneo and MSCVpac.

EXAMPLE 2

[0039] Analysis of density-dependent growth. The NIH 3T3 fibroblastswere infected with helper-free retrovirus encoding wild-type andinducible versions of E2a-Pbx1. Stably expressing cells, as well ascells transduced with empty vector virus, were selected 7 days in 1mg/ml G418 in the absence of estrogen. Equivalent numbers of cells wereplated in triplicate into 60-mm dishes in the presence and absence of 1μM estradiol. Half media changes were performed every two days and thetotal number of live, adherent cells was determined after 14 days. Folddensity was calculated in comparison to the number of cells transducedwith the empty viral vector.

EXAMPLE 3

[0040] Retroviral infection of primary murine marrow progenitor cells.Bone marrow mononuclear cells were isolated on a Ficoll-Paque gradientfollowing harvest from the femurs and tibia of female Balb/c miceinjected intraperitonally with 5-fluorouacil (150 mg/kg) 5 days prior toharvest. Marrow progenitor cells were purified by negative selection oflineage-positive cells on a magnetic column with a murine progenitorcell antibody cocktail (Stemcell Technologies, Vancouver, BC, Canada).Progenitor cells were prestimulated for 48 hours in Iscoves modifiedDulbecco medium (IMDM) containing 15% fetal bovine serum (FBS), 50 ng/mlstem cell factor (SCF), 25 ng/ml IL-3 and 25 ng/ml IL-6.

[0041] Helper-free retrovirus was prepared by calcium phosphatetransfection of 293T cells with MSCV retroviral constructs and anecotropic packaging construct. Then, 25,000 marrow progenitor cells wereinfected with 1 ml retroviral supernatant (approximate titer 2×10⁵particles/ml) by spinoculation (2500 g, 2 hours, 22° C.) in the presenceof Lipofectamine (1:1000, Gibco BRL). Following spinoculation, the cellswere cultured in RPMI media containing GM-CSF and 1 μM β-estradiol, anestrogen analog, as described above. Immortalized myeloid progenitorcells were enriched by the passage of non-adherent cells over the courseof approximately 3 weeks.

[0042] Single cell clones were prepared by limiting dilution.E2a-Pbx1mediated conditional Myeloid (ECoM)-GM and ECoM-G clones wereestablished from both populations immortalized by EP Δ578ER and EPΔ623ER, whereas the ECoM-M cell line was derived from a populationimmortalized by EP Δ623ER.

EXAMPLE 4

[0043] Phagocytosis assay. Phagocytosis was assayed using fluoresceinisothiocyanate (FITC)-labeled heat killed Escherichia coli BioParticles(Molecular Probes, Eugene, Oreg.). The 10⁷ BioParticles and 10⁶ cellswere incubated for 1 hour at 37° C. with shaking. Followingcytocentrifugation onto Superfrost Plus slides (Fisher, Pittsburgh,Pa.), the nuclei were counterstained with bis-Benzimide (Hoechst 33258,Sigma) and the F-actin counterstained with TRITC-phalloidin (Sigma).Images were captured with DeltaVision deconvolutuion microscope systemand the data sets were deconvoluted and analyzed using Soft Worxsoftware (both from Applied Precision, Issaquah, Wash.).

EXAMPLE 5

[0044] Flow cytometric analysis. The FITC-labeled monoclonal antibodiesGR-1 (ly6G) and Mac-1 (CD11b) were purchased from Pharmagen and F4/80from Serotec. Then, 10⁶ cells were labeled 30 minutes at 4° C. inphosphate-buffered saline (PBS)/1% FBS/0.1% NaN₃, washed, andresuspended in the same buffer with 2 μg/ml propidium iodide (PI). Flowcytometry data were acquired with the program CELLQuest on a bench-topflow cytometer. Live cells were gated for analysis by forward and sidescatter for lack of PI staining.

EXAMPLE 6

[0045] Retroviral infection of ECoM-G cells with heterologousoncoproteins. Helper-free retrovirus was generated for wild-typeE2a-Pbx1, Hoxa7, Hoxa9, Nup98/HoxA9, Hoxb8, AML1/ETO, PML/RARα andPLZF/RARα constructs cloned into the multiple cloning site of theMSCVneo and MSCVpac retroviral vectors. ECoM-G cells (250,000) from 3different clones were transduced by spinoculation (as described above)and selected 5 days in G418 (1 mg/ml) or puromycin (1 μg/ml). The cellswere washed and plated in media without estrogen. Only those cells thatwere capable of continued and indefinite proliferation in the absence ofestrogen were expanded to generate RNA for Northern analysis.

EXAMPLE 7

[0046] Analysis of differentiation arrest induced by heterologousoncoproteins in E2a-Pbx1-ER expressing cells. The ECoM cells provide asystem in which to dissect the mechanisms of action of oncogenes.Heterologous oncoproteins were introduced into ECoM-G clones byretroviral infection. Expression was verified by immunoblot assay andcells were assayed for the ability to re-establish differentiationarrest following estrogen withdrawal. AML1/ETO, PML/RARα, Hoxa9, Hoxb8and wild type E2a-Pbx1 prevented granulocytic differentiation ofspecific clones permitting their continued and indefinite proliferationin the presence of GM-CSF. The resultant pattern of gene expression wasconsistent with differentiation arrest by the new oncoprotein. Hoxa9,for example, permitted up-regulation of NE, gp91^(PHOX), Ly6G and theG-CSF-R to levels similar to those of primary marrow immortalized by theexpression of Hox9a alone. Similarly, whereas AML1/ETO permittedup-regulation of gp91^(PHOX) and Ly6G, the expression of NE and G-CSF-R,whose activation requires AML1 remain low. This pattern is consistentwith the dominant negative function of AML1/ETO on AML1 responsivepromoters. ECoM-G clones arrested in differentiation by PML/RARα orPLZF/RARα showed RA sensitivity not seen in the parental cells,providing mechanistic evidence of the function of the secondoncoprotein. Although parental cells were completely unresponsive totreatment with 10 μM atRA for 4 days, both PML/RARα and PLZF/RARαderivatives stopped proliferating and underwent quantitativegranulocytic differentiation. The ECOM cell lines can thus be used forthe analysis of the biochemical and genetic mechanisms by whichoncoproteins prevent stage specific myeloid differentiation.

EXAMPLE 8

[0047] Assay for oncogenic activity of known or unknown proteins. TheEcoM cells provide a system in which to examine suspected oncoproteinsand their effects on granulocytic differentiation. Both Nup98/HoxA9 andHoxa7 were capable of arresting ECoM-G cell differentiation followingestrogen withdrawal. Neither protein was known to function in thiscapacity. Despite their equivalent levels of expression, Nup98/HoxA9 wassignificantly less efficient than either Hoxa7 or Hoxa9 in arrestingdifferentiation precipitated by estrogen withdrawal. This suggested thatthe mechanism of differentiation arrest by Nup98/HoxA9 was fundamentallydifferent from that of Hoxa7 or Hoxa9. The small fraction (<1%) ofECoM-G cells that continued to proliferate expressed a smaller versionof the protein by western analysis, indicating that some form ofrearrangement had occurred. Nup98/HoxA9, identified as the product ofthe t(7; 11) translocation in human myeloid leukemia has also been shownto cause transformation in NIH 3T3 fibroblasts and to cause AML in mice.The pattern of gene expression in Nup98/HoxA9-immortalized ECoM-G cellswas similar to that in Hoxa9-immortalized cells, but lacking gp91^(PHOX)expression. Such an experiment could be performed to determine theoncogenicity of a protein or polypeptide by inserting a cDNA into theappropriate viral vector, infecting the cells, assaying for expression,and observing the proliferative capacity of the cells in the absence ofestrogen.

EXAMPLE 9

[0048] Analysis of cooperativity between E2a-Pbx1-ER and activatedoncogenes. The ECoM-G and ECoM-M clones were rendered GM-CSF-independentfollowing infection with retrovirus encoding oncogenic H-Ras^(L61) orBcr/Abl^(p190). Removal of estrogen from these cells permitteddifferentiation as assessed by Wright-Giemsa staining. Although theparental EcoM-G cells in the presence of GM-CSF underwent strictgranulocytic differentiation (98%), ECoM-G-Ras and ECoM-G-Bcr/Abl cellsdemonstrated a predominant monocytic differentiation (99% and 65%respectively). In contrast, the differentiation of ECoM-M cells was notmorphologically affected by expression of Ras or Bcr/Abl, though maturemonocytes expressing Ras were no longer adherent. These morphologicalresults were supported by flow cytometric analysis. Differentiation ofECoM-G cells expressing Ras or Bcr/Abl was accompanied increased F4/80and decreased GR-1 staining, whereas expression of Ras or Bcr/Abl inECoM-M cells did not significantly alter GR-1, MAC-1 or F4/80 staining.

[0049] These data clearly demonstrate the advantage of the instantinvention over the use of cells that have been in culture for extendedperiods of time that contain unknown genetic alterations. It is clearthat the oncogene present and activated have a large affect on the stateof differentiation of the cells. By controlling the factors expressed inthe cell, one is able to dissect the mechanisms of differentiation andleukemogenesis.

EXAMPLE 10

[0050] Assay for chemotherapeutic activity of a compound. A panel ofheterologous oncoproteins are introduced into ECoM-G clones byretroviral infection and stable daughter lines are established.Expression is verified by immunoblot assay and cells were assayed forthe ability to re-establish differentiation arrest following estrogenwithdrawal. AML1/ETO, PML/RARα, Hoxa9, Hoxb8and wild type E2a-Pbx1 areall capable of preventing granulocytic differentiation of specificclones permitting their continued and indefinite proliferation in thepresence of GM-CSF. Cells expressing each of the different oncogenes isplated into a 96- or 384-well plate. The cells are exposed to a seriesof compounds derived from any of a number of sources. Cells are observedto determine if they undergo differentiation and assayed for functional(e.g. NADPH oxidase activity) or expression of cell surface markers ofdifferentiation (e.g. upregulation of GR-1). Compounds that causedifferentiation are further analyzed.

[0051] Although an exemplary embodiment of the invention has beendescribed above by way of example only, it will be understood by thoseskilled in the field that modifications may be made to the disclosedembodiment without departing from the scope of the invention, which isdefined by the appended claims.

We claim:
 1. A cDNA sequence encoding a hormone sensitiveE2a-Pbx1protein comprising a nucleotide sequence encoding a chimericE2a-Pbx1-hormone binding domain (HBD) protein.
 2. A cDNA sequence ofclaim 1, wherein the HBD is inserted downstream of the E2a codingsequence and upstream of a DNA-binding homeodomain of the Pbx1 codingsequence.
 3. The cDNA sequence of claim 1, wherein the HBD is derivedfrom a hormone receptor selected from the group consisting of estrogenreceptor (ER), glucocorticoid receptor (GR), thyroid hormone receptor(THR), mineralocorticoid receptor (MR), androgen receptor (AR), andprogesterone receptor (PR).
 4. The cDNA sequence of claim 1, wherein thecoding sequence of the HBD contains a mutation to decrease thesensitivity of the domain to hormone.
 5. A protein encoded by the cDNAof claim
 1. 6. A cell expressing the cDNA of claim
 1. 7. Ahormone-dependent E2a-Pbx1hematopoietic cell line comprising primarymammalian hematopoietic progenitor cells expressing a hormone dependentE2a-Pbx1protein expressing an E2a-Pbx1protein fused to a HBD.
 8. Thehormone-dependent E2a-Pbx1hematopoietic cell line of claim 7, whereinthe HBD is derived from a hormone receptor selected from the groupconsisting of estrogen receptor (ER), glucocorticoid receptor (GR),thyroid hormone receptor (THR), mineralocorticoid receptor (MR),androgen receptor (AR), and progesterone receptor (PR).
 9. Thehormone-dependent E2a-Pbx1hematopoietic cell line of claim 7, whereinthe HBD is inserted downstream of the E2a coding sequence and upstreamof a DNA-binding homeodomain of the Pbx1 coding sequence.
 10. Thehormone-dependent E2a-Pbx1hematopoietic cell line of claim 7, whereinthe coding sequence of the HBD contains a mutation to decrease thesensitivity of the domain to hormone.
 11. The hormone-dependentE2a-Pbx1hematopoietic cell line of claim 7, wherein the cells undergodifferentiation upon withdrawal of hormone.
 12. A method for screeningfor chemotherapeutic factors comprising exposing a hormone-dependentE2a-Pbx1hematopoietic cell line to its appropriate hormone, expressing aheterologous oncogene within the cells, withdrawing hormone from thecells, exposing the cells to the potential chemotherapeutic factor, anddetermining if the cells undergo differentiation.
 13. The method ofclaim 12, wherein the hormone-dependent E2a-Pbx1 hematopoietic cell linecomprises primary mammalian hematopoietic progenitor cells expressing ahormone-dependent E2a-Pbx1protein expressing an E2a-Pbx1protein fused toa HBD.
 14. The method of claim 12, wherein the HBD is inserteddownstream of the E2a coding sequence and upstream of a DNA-bindinghomeodomain of the Pbx1 coding sequence.
 15. The method of claim 12,wherein the HBD is derived from a hormone receptor selected from thegroup consisting of estrogen receptor (ER), glucocorticoid receptor(GR), thyroid hormone receptor (THR), mineralocorticoid receptor (MR),androgen receptor (AR), and progesterone receptor (PR).
 16. The methodof claim 12, wherein the hormone binding domain contains a mutation todecrease the sensitivity of the domain to hormone.